Process for producing purified anthocyanin and crystalline anthocyanin

ABSTRACT

Provided are a process for producing purified anthocyanidin glucoside in which a rhamnose end of anthocyanidin rutinoside is cleaved using rhamnosidase to convert the anthocyanidin rutinoside component into anthocyanidin glucoside, the anthocyanidin glucoside component being then purified and isolated; or a crystalline anthocyanidin glucoside salt obtained by further crystallizing the purified anthocyanidin glucoside and a process for producing the same. 
     Also provided are a process for producing purified anthocyanidin rutinoside in which a glucose end of anthocyanidin glucoside is cleaved using β-glucosidase to degrade and remove the end, the anthocyanidin rutinoside component being then purified and isolated; or a crystalline anthocyanidin rutinoside salt obtained by further crystallizing the purified anthocyanidin rutinoside and a process for producing the same.

TECHNICAL FIELD

The present invention relates to a process for producing purifiedanthocyanin from anthocyanin derived from natural product and a processfor producing crystalline anthocyanin by further crystallizing purifiedanthocyanin, and a crystalline anthocyanin prepared by theaforementioned process.

More particularly, the present invention relates to a process whichfacilitates the subsequent purification and crystallization steps byenzymatically converting or removing anthocyanidin rutinoside oranthocyanidin glucoside constituting anthocyanins to decreaseanthocyanidin rutinoside or anthocyanidin glucoside.

BACKGROUND ART

Anthocyan is a generic term for anthocyanidin, which has a backbonerepresented by the following formula (I), in combination withanthocyanin, which is a glycoside formed by binding of saccharide toanthocyanidin.

(I)

R¹ R² delphinidin OH OH cyanidin OH H malvidin OCH₃ OCH₃ pelargonidin HH peonidin OCH₃ H petunidin OCH₃ OH

Examples of anthocyanidin, i.e., an aglycon, include delphinidin,cyanidin, malvidin, pelargonidin, peonidin, and petunidin. Anthocyaninis referred to as anthocyanidin glucoside when, for example, glucose isbound to the anthocyanidin as a glycoside. Saccharide found inanthocyanin includes: monosaccharide such as galactose and arabinose inaddition to glucose; and disaccharide such as rutinose and sophorose.

Anthocyans are widely present in nature, and are mainly used as anatural pigment for food or, because of their functionalities, areextensively used for pharmaceuticals, quasi drugs, cosmetics, and thelike in Europe. For example, use thereof as a cicatrizant, as disclosedin Japanese Examined Patent Publication (Kokoku) No. 59-53883, orpharmacological properties thereof which are valuable in the treatmentof peripheral blood diseases using anthocyanin derived from blueberry,as disclosed in Japanese Laid-open Patent Publication (Kokai) No.3-81220, have been discovered. In recent years, the functionality ofanthocyanin has drawn attention in Japan for uses of anthocyanin otherthan as a pigment. The present inventors have also found several usefulefficacies in anthocyanin of black currant and these are reported in WO01/01798.

When these anthocyanins having pharmacological properties are used aspharmaceuticals and the like, highly purified ones are required.Heretofore, however, mass production of highly purified anthocyanins hasnever been realized. Further, while highly purified anthocyanins arepreferably crystalline from the viewpoint of stability, hygroscopicity,and the like, mass production of crystalline anthocyanins has likewisenot been realized up to now.

Conventionally, anthocyanin compositions for pharmaceuticals are mainlypreparations derived from blueberry with an anthocyanin content of 25%by weight or lower. Thus, at least several hundred mg of an anthocyaninpreparation had to be administered per dose in order to exhibit itseffectiveness, and the consumption of a small amount thereof could notproduce pharmacological effects in practice. Accordingly, compositionscontaining highly purified anthocyanin at high levels have been awaited.

Highly purified anthocyanin was not present because of the followingreasons. For example, in the case of blueberry-derived anthocyanin,there are 15 types of anthocyanin components, and the physiochemicalproperties of these substances are very similar to one another. Thus,the respective flux peaks thereof overlap with one another inpurification using a preparative column or the like. Or, separation andpurification were impossible because each component was in a very smallamount.

In the case of anthocyanin derived from black currant, for example, fourcomponents, i.e., cyanidin-3-O-glucoside (hereinafter it is abbreviatedto “C3G”), cyanidin-3-O-rutinoside (hereinafter it is abbreviated to“C3R”), delphinidin-3-O-glucoside (hereinafter it is abbreviated to“D3G”), and delphinidin-3-O-rutinoside (hereinafter it is abbreviated to“D3R”), are contained as anthocyanins. As with the blueberry-derivedanthocyanin, due to very similar physiochemical properties among thefour substances, even mixtures of these four components have very closechromatography peaks. Even if preparative chromatography or centrifugalpartition chromatography were performed to obtain purified anthocyanin,mass production thereof was impossible due to extremely deterioratedyield. The quantitative ratio of representative anthocyanins in blackcurrant is as follows: D3G, D3R, C3G, and C3R are respectively presentat 12.5%, 47.9%, 4.1%, and 35.5%. Consequently, purification of a largeamount of D3G and C3G components, which are contained at low levels,involved further difficultly, and thus a process for mass producingpurified anthocyanin has been awaited.

In contrast, anthocyan has been heretofore known to have a drawback inits stability. The present inventors have applied for patent on aprocess for stabilizing substances containing anthocyanin at high levelby adding phytic acid, saccharides, and sugar alcohols as stabilizers(PCT/JP00/09204). However, when a large amount of anthocyanin is usedfor making preparations, there is no room to contain these additives.Accordingly, more stable physical properties were required andpreparations using crystalline anthocyanin, which is physically morestable, were awaited. Thus, conventionally, high purity anthocyanin wasorganically synthesized for pharmaceutical applications through manysteps by, for example, a process for synthesizing delphinidinhydrochloride (anthocyanidin hydrochloride of an aglycon instead ofglycoside) as disclosed in Japanese Patent No. 3030509, although massproduction thereof from natural products was not realized.

The anthocyanidin hydrochloride produced by the synthesis method isstable under strong acidic conditions, however, it is likely to bedegraded as compared to a glycoside in weak acidic to neutral regions.The application range was thus very narrow. Accordingly, production ofanthocyanin which was more stable in acidic to neutral regions ascrystals was awaited, although mass production of anthocyanin by theorganic synthesis process is currently still unavailable.

Features of anthocyanins are listed in the Dictionary of NaturalProducts (issued by Chapman & Hall, 1994, London). For example, thecrystal of D3R has not been heretofore reported, and while the crystalform of D3G hydrochloride has been reported, its melting point is notdescribed. This indicates that mass production thereof was difficult.Similarly, although the melting point and the crystal form of C3Rhydrochloride are described, there is no description on the meltingpoint of C3G hydrochloride. This indicates that mass production thereofwas also difficult. Up to the present, only a very small amount ofpurified anthocyanin could be produced, regardless of whether it iscrystalline or not. Thus, there was substantially no study whichinvestigated the reactivity of various enzymes to anthocyanin.Especially, there was no description on the reactivity of rhamnosidaseto anthocyanin. Accordingly, a process for mass producing highlypurified anthocyanin from natural products without using complicatedsynthesis processes has been awaited. Further, a process for massproducing crystalline anthocyanin salts by crystallizing purifiedanthocyanin was also awaited.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a process for producingpurified anthocyanin in which a rhamnose end of anthocyanidin rutinosideis cleaved using rhamnosidase to convert the anthocyanidin rutinosidecomponent into anthocyanidin glucoside, the anthocyanidin glucosidecomponent being then purified and isolated, or a process for producingcrystalline anthocyanidin glucoside salt hydrate by furthercrystallizing the purified anthocyanin.

Another aspect of the present invention relates to a process forproducing purified anthocyanin in which a glucose end of anthocyanidinglucoside is cleaved using β-glucosidase to degrade and remove the end,the anthocyanidin rutinoside component being then purified and isolated,or a process for producing crystalline anthocyanidin rutinoside salthydrate by further crystallizing the purified anthocyanin.

A further aspect of the present invention relates to a crystallineanthocyanin salt hydrate prepared by these production processes.

More specifically, the first invention provides a process for producingpurified anthocyanidin glucoside, wherein rhamnosidase is allowed to actupon an anthocyanin composition containing at least one kind ofanthocyanidin rutinoside, and anthocyanidin rutinoside is subjected tohydrolysis to convert into anthocyanidin glucoside, which is thenisolated and purified.

The second invention provide a process for producing purifiedanthocyanidin rutinoside, wherein β-glucosidase is allowed to act uponan anthocyanin composition containing at least one kind of anthocyanidinglucoside and anthocyanidin rutinoside, and anthocyanidin glucoside issubjected to hydrolysis to reduce the anthocyanidin glucoside, theanthocyanidin rutinoside being then isolated and purified.

In the aforementioned first and second inventions, the anthocyanincomposition includes fruit juice obtained from at least one memberselected from black currant, fig, coffee, banana, blackberry, and thelike, and/or an anthocyanin concentrate obtained from wild rice (Zizaniaaquatica Linn.), colocasia, and the like. The rhamnosidase includeshesperidinase, naringinase and the like. Further, the β-glucosidase canselectively degrade only the β-glucoside bond of anthocyanidin glucosidewithout degrading the β-glucoside bond of anthocyanidin rutinoside, andspecific examples thereof include β-glucosidase derived from almond.

The third invention provides a process for producing crystallineanthocyanidin-3-O-glucoside hydrochloride hydrate through the steps of:

a) allowing rhamnosidase to act upon an anthocyanin compositioncontaining at least one kind of anthocyanidin rutinoside and subjectinganthocyanidin rutinoside to hydrolysis to convert into anthocyanidinglucoside;

b) purifying the anthocyanidin glucoside to obtain anthocyanidinglucoside of 99% or higher purity; and

c) crystallizing the anthocyanidin glucoside using a mixed solvent ofhydrochloric acid/alcohol system.

The fourth invention provides a crystalline anthocyanidin-3-O-glucosidehydrochloride hydrate obtained by the method according to the thirdinvention.

The fifth invention provides a process for producing crystallineanthocyanidin-3-0rutinoside hydrochloride hydrate through the steps of:

a) allowing β-glucosidase to act upon an anthocyanin compositioncontaining at least one kind of anthocyanidin glucoside andanthocyanidin rutinoside, and subjecting anthocyanidin glucoside tohydrolysis to reduce the anthocyanidin glucoside;

b) purifying the anthocyanidin rutinoside to obtain anthocyanidinrutinoside of 99% or higher purity; and

c) crystallizing the anthocyanidin rutinoside using a mixed solvent ofhydrochloric acid/alcohol system.

The sixth invention provides a crystalline anthocyanidin-3-O-rutinosidehydrochloride hydrate obtained by the method according to the fifthinvention.

In the third and the fifth inventions, the purification processaccording to step b) can be carried out by ion exchange adsorptionchromatography and/or HPLC, and the mixed solvent of hydrochloricacid/alcohol system used in step c) can be composed of 5% (v/v)hydrochloric acid/95% (v/v) methanol.

The seventh invention provides a crystalline delphinidin-3-O-glucosidehydrochloride 0.5 hydrate having the following physical properties:

Melting point based on thermoanalysis: 258° C. Uvλ max(ε): 517 nm(27500) FAB-MS m/z:M⁺:465

Compositional formula: C₂₁H₂₁O₁₂Cl•0.5H₂O Elementary analysis: C H ClMeasured values: 48.00 4.50 6.80

The eighth invention provides a crystalline cyanidin-3-O-glucosidehydrochloride 0.5 hydrate having the following physical properties:

Melting point based on thermoanalysis: 245° C. Uvλ max(ε): 510 nm(26300) FAB-MS m/z:M⁺:449

Compositional formula: C₂₁H₂₁O₁₁Cl•0.5H₂O Elementary analysis: C H ClMeasured values: 48.80 4.70 6.90

The ninth invention provides a crystalline delphinidin-3-O-rutinosidehydrochloride 1.5 hydrate having the following physical properties:

Melting point based on thermoanalysis: 224° C. Uvλ max(ε):520 nm (27800)FAB-MS m/z M⁺:611

Compositional formula: C₂₇H₃₁O₁₆Cl•1.5H₂O Elementary analysis: C H ClMeasured values: 45.80 5.30 5.20

The tenth invention provides a crystalline cyanidin-3-O-rutinosidehydrochloride 0.5 hydrate having the following physical properties:

Melting point based on thermoanalysis: 214 to 226° C. Uvλ max(ε):512 nm(27400) FAB-MS m/z: M⁺:595

Compositional formula: C₂₇H₃₁O₁₅Cl•0.5H₂O Elementary analysis: C H ClMeasured values: 50.00 5.30 5.30

The term “anthocyanin” used herein refers to a glycoside which wasprepared by the binding of saccharides to anthocyanidin, i.e., anaglycon, and examples thereof include glucoside, rutinoside,arabinoside, and galactoside to which saccharides such as glucose,rutinose, arabinose, and galactose have been bonded.

In the present invention, anthocyanin compositions containing at leastone kind of anthocyanidin glucoside and/or anthocyanidin rutinoside thatare used in the enzyme reaction may be any substances which containanthocyanin. Examples thereof include commercially available fruitjuice, concentrated juice, beverages, pigment solutions, and powderswhich are starting materials for foods, pharmaceuticals, or the like.Preferably, concentrated juice is fruit juice obtained from at least onemember selected from black currant, fig, coffee, banana, blackberry, andthe like, and/or an anthocyanin concentrate which is obtained from wildrice (Zizania aquatica Linn.), colocasia, and the like. The powders arefirst dissolved in water, buffer, or the like to use the solution in thereaction. More preferably, compositions containing anthocyanin at highlevels which are prepared by the method reported in WO 01/01798 by thepresent inventors are used. Since acids, saccharides, polyphenols, andthe like are previously removed, operation in the purification stepafter the enzyme reaction is facilitated.

The types of aglycon of anthocyanin used for the process for producingpurified anthocyanin according to the present invention are notparticularly limited. Black currant-derived anthocyanins and saltsthereof are preferred. An aglycon moiety of the black currant-derivedanthocyanin consists only of delphinidin and cyanidin. In the enzymereaction, however, these two types are not considered to be particularlyspecific for anthocyanidin, and can be applied to a glycoside of anotheranthocyanidin, for example, glycosides such as malvidin, pelargonidin,petunidin, and peonidin represented by formula (I). Further, the blackcurrant-derived anthocyanin component is constituted by four components:cyanidin-3-O-glucoside (C3G), cyanidin-3-O-rutinoside (C3R),delphinidin-3-O-glucoside (D3G), and delphinidin-3-O-rutinoside (D3R).

The types of salts which constitute crystalline anthocyanin salt includesalts with mineral acids such as hydrochloric acid and sulfuric acid andsalts with organic acids (flavinium salt) such as phosphoric acid,tritluoroacetic acid (TFA), and acetic acid. From the viewpoint of easycrystallization, salts with hydrochloric acid, TFA, and phosphoric acidare preferable.

Rhamnosidase that is used in the present invention is not particularlylimited, and it may originate from animals, plants, microorganisms, andthe like as long as it has α-rhamnosidase activities at the anthocyaninend as the action mechanism. However, it is important that it has lowβ-glucosidase activities. Further, anthocyanin is known to be degradedwhen it is allowed to stand in neutral to basic regions for a longperiod of time, and its degradation is accelerated by heating.Accordingly, it is necessary that the activity level is high at pH 4.0or below and at 40° C. or below. Preferable examples thereof includehesperidinase or naringinase, with hesperidinase being more preferred.Hesperidinase includes Aspergillus niger-derived substances which areindustrially produced for the purpose of degrading hesperidin, which isan opaque white component of Satsuma mandarins. The reactivity ofAspergillus niger-derived hesperidinase to anthocyanin has not beenheretofore known. Naringinase also includes Aspergillus niger-derivedsubstances which are used for taste improvement by degrading naringin,which is a bitter principle from abscised oranges or Citrus aurantium(natsu-mikan). It should be noted that the application of Aspergillusniger-derived naringinase to anthocyanin has not been reported.

The source of the β-glucosidase that is used in the present invention isnot particularly limited. It must react with anthocyanin and it shouldhave a substrate specificity to degrade only the β-glucoside bond ofanthocyanidin glucoside without degrading the β-glucoside bond ofanthocyanidin rutinoside. Specifically, suitable β-glucosidase reactswith only terminal glucose but it does not react with glucose which ispresent at the center in the molecule. As with rhamnosidase, theactivity level should be high at pH 4.0 or below and at 40° C. or below,and preferable examples thereof include almond-derived β-glucosidase.While the almond-derived β-glucosidase is a representativeβ-glucosidase, there has been no study which investigated the reactivityto anthocyanin.

Examples of treatments utilizing the reaction of β-glucosidase toanthocyanin include the conventional use of anthocyanase, which is atype of β-glucosidase, for decolorization and debittering in the fruitjuice industry for grape juice and peach nectar and the like, and in thewine industry for wines and sparkling wines including champagnes.However, anthocyanase reacts with and degrades both glucoside andrutinoside of anthocyanin. Thus, it is not suitable for the productionof purified anthocyanin.

The reaction conditions which allow these enzymes to act on anthocyaninare not particularly limited. As described above, anthocyanin is knownto be degraded when it is allowed to stand in neutral to basic regionsfor a long period of time and its degradation is accelerated by hightemperature. Thus, an acidic region is preferable, and preferably, it isnot reacted at high temperature. Specifically, reaction at pH 4.0 orbelow and at 40° C. or below is likely to be most preferable.

The substrate concentration in the anthocyanin-containing substance thatis used in the enzyme reaction is not particularly limited. When theconcentration is extremely high, the viscosity of the reaction solutionincreases and the reaction speed is lowered. Due to a fear of inhibitoryreaction by substances in the reaction solution or transition reactionto a substrate, the substrate concentration is preferably 10% (w/v) orbelow, and more preferably 5% (w/v) or below. The amount of enzymesadded is not particularly limited in relation to the reaction time.

After the content of a substrate in the anthocyanin-containingsubstance, i.e., anthocyanidin rutinoside or anthocyanidin glucoside, isdecreased by the enzyme reaction using rhamnosidase or β-glucosidase,the enzyme reaction can be terminated by commonly employed methods, andexamples thereof include raising the temperature by heating, pHadjustment by adding acid or alkali, and addition of organic solvent. Asdescribed above, anthocyanins are unstable in neutral to basic regionsand their degradation is accelerated by heating. Thus, the enzymereaction is preferably terminated by pH lowering with the addition ofstrong acids such as hydrochloric acid, TFA, and phosphoric acid or bythe addition of organic solvent such as methanol.

In the present invention, purification after the completion of theenzyme reaction can be carried out by column chromatography as well asby a suitable combination of another chromatography, resin adsorption,membrane separation, and the like, if necessary. Of all the methods,chromatography using ODS-silica gel is most preferable. If necessary,the anthocyanin component may be first adsorbed on a cation exchangeresin or the like and then eluted in order to similarly purify theprepurified one.

The anthocyanin content herein is determined by summing the contents ofeach of the anthocyanin components contained, which are calculated bythe peak area ratio of anthocyanin by HPLC, as with the disclosure inPCT/JP 00/09204 and WO 01/01798.

Specifically, an anthocyanin sample with a known weight is firstanalyzed by HPLC and the calibration curve is prepared based on the peakarea at 520 nm, to thereby determine the content of each anthocyanincomponent. Further, the content of each of the components is divided bythe peak area to determine the response coefficient, i.e., the mg/peakarea. Subsequently, the anthocyanin-containing sample is subjected toHPLC analysis, the response coefficient which was determined from thesample is multiplied by the peak area of each component to calculate thecontent of each component. Thus, the anthocyanin purity is determined as% by weight (w/w) based on the ratio with the amount injected.Accordingly, the purity of anthocyanin is calculated by including theamount of a bound saccharide in addition to the amount of anthocyanidin,i.e., an aglycon. The purity level of the purified anthocyanin accordingto the present invention is 99% or higher based on the HPLC analysis.

The crystallization process according to the present invention ispreferably carried out mainly from an organic solvent, and methanol ispreferably used as the crystallizing solvent. Because anthocyanins arecrystallized by generating salts with acids, the addition of acids inthe crystallizing solvent is preferred. Preferably, about 1% to 5% (v/v)of hydrochloric acid, TFA, phosphoric acid, or the like is added.

The crystalline D3C, crystalline D3R, crystalline C3G, and crystallineC3R which were prepared in Example 1 or 3 below were subjected tothermoanalysis, and the results thereof are provided below.

The melting point of D3G is 258° C., that of D3R is 224° C., that of C3Gis 245° C., and that of C3R is 214 to 226° C.

The crystalline anthocyanin salt according to the present invention iscrystal having 99% or higher purity based on the polarizationmicroscopy, elementary analysis, melting point determination, and HPLCanalysis, and is a very stable substance without hygroscopicity and witha melting point of 200° C. or higher.

In the past, highly purified anthocyanin or crystalline anthocyanin saltcould not be produced. With the use of the production process accordingto the present invention, however, highly purified anthocyanin andcrystalline anthocyanin salt could be produced through purification fromnatural products. The thus produced crystalline anthocyanin salts didnot exhibit any hygroscopicity and were stable.

BEST MODE FOR CARRING OUT THE INVENTION

The present invention will be described in more detail with reference tothe following examples, reference examples, and comparative examples.The technical scope of the present invention, however, is not limited bythese examples.

REFERENCE EXAMPLE 1 Preparation of Composition Containing Anthocyanin atHigh Level

In accordance with the process as described in WO 01/01798, compositionscontaining anthocyanin at high level were prepared. Specifically, 3 kgof commercially available concentrated black currant juice (theanthocyanin purity per solid content: 2.8%) was diluted with water toprepare diluted fruit juice with a concentration of Bx 10. The dilutedfruit juice was filtered with a filter paper to remove foreign matter.Thereafter, membrane separation was carried out using a membraneseparator (NTR-7410, Nitto Denko Co., Ltd.). The concentrated liquidobtained by membrane separation was spray dried to obtain a powderycomposition containing anthocyanin at high level. The anthocyanin purityof this composition was 14.1% per solid content. This compositionexhibited hygroscopicity when it was allowed to stand at roomtemperature.

EXAMPLE 1 Production of Purified delphinidin-3-O-glucoside and Purifiedcyanidin-3-O-glucoside Using Hesperidinase

The powder obtained in Reference Example 1 (40 g) (anthocyanin purity:14.1%; proportion of each anthocyanin component: 12.5%, 47.9%, 4.1%, and35.5% for D3G, D3R, C3G, and C3R, respectively) was dissolved in 1 literof 50 mM acetate buffer (pH 3.5) to prepare an anthocyanin substratesolution. The calculated anthocyanin content in the substrate solutionwas 5.64 g, and the contents of D3G, D3R, C3G, and C3R were 0.71 g, 2.70g, 0.23 g, and 2.00 respectively.

Separately, 79.35 g of hesperidinase (tradename: Hesperidinase Tanabe 2,Tanabe Seiyaku Co,. Ltd) was dissolved in 1 liter of 50 mM acetatebuffer (pH 3.5) to prepare an enzyme solution (corresponding to therhamnosidase activity of 42.5 U/ml).

The substrate solution and the enzyme solution were respectively heatedat 40° C. and were then mixed to initiate the reaction. The reaction wascarried out at 40° C. for 6 hours, and 2 liters of 3% (w/v) phosphoricacid solution was added to terminate the reaction. Subsequently, 4liters of ion exchange resin XAD-7 (Rohm and Haas Company) was filledinto a column (13 cm (inside diameter)×30 cm (length)), and the reactionmixture (4 liters) was passed therethrough to allow the anthocyanincomponent to adsorb thereon. Subsequently, 0.1% (w/v) TFA (2 liters) wasallowed to pass therethrough to wash the nonadsorbed component. Further,an 80% (v/v) aqueous methanol solution containing 0.1% TFA was passedtherethrough to eluate the adsorbed component. This methanol solutionwas concentrated using a rotary evaporator and converted into an aqueous3% phosphoric acid solution to bring the concentration of the solidcontent to 10% (w/v). Thus, a concentrated liquid was obtained.

This concentrated liquid was detected using an ODS-120T silica gelcolumn (ED 5.5×30 cm, 20 μm, TOSOH CORPORATION) with an aqueous 9% (v/v)acetonitrile solution containing 0.1% TFA at a flow rate of 80 ml/min ata wavelength of 520 nm to obtain the D3G fraction with a retention time(R.T.) of 66 to 90 min and the C3G fraction with an R.T. of 158 to 200min. These fractions has a single peak based on the HPLC analysis, andpurified delphinidin-3-O-glucoside and purified cyanidin-3-O-glucosidewith a purity level of 99% or higher could be obtained.

Conditions for the HPLC analysis to measure the purity are as follows.Specifically, the analysis was performed under the following gradientconditions using the Hewlett Packard Series 1100 HPLC System (YokogawaAnalytical Systems Inc.).

HPLC Gradient Conditions:

Liquid A (aqueous 0.5% Time (min) phosphoric acid solution) Liquid B(methanol) 0 80 20 15 77 23 20 77 23 30 50 50 40 50 50

A Zorbax SB-C18 column (4.6 mm×250 nm, 5 μm, Hewlett Packard) was used.Detection was carried out at the flow rate of 1 ml/min at the wavelengthof 520 nm. The R.T.s of the sample D3G and C3G were respectively 10.54min and 14.60 min.

A part of the substrate solution before the enzyme reaction and a partof the solution in which the enzyme reaction was terminated werecollected, and foreign matters were removed through a microfilter with apore diameter of 0.45 μm to prepare an anthocyanidin glucoside solution.The contents of anthocyanin components before the reaction and after thereaction were measured. The results are as shown in Table 1. The resultsindicate that the substrate solution consisted only of anthocyanidinglucoside because anthocyanidin rutinoside was degraded andanthocyanidin glucoside was generated in the substrate solution. Inaddition, the amount of D3G was 3.36 times higher than before thereaction, and the amount of C3G was 6.78 times higher than before thereaction.

TABLE 1 Change in anthocyanin content D3G D3R C3G C3R Before reaction0.71 g 2.70 g 0.23 g 2.00 g After reaction 2.39 g 0.00 g 1.56 g 0.00 g

EXAMPLE 2 Production of Crystalline delphinidin-3-O-glucosideHydrochloride Hydrate and Crystalline cyanidin-3-O-glucosidehydrochloride Hydrate

The D3G fraction and C3G fraction obtained in Example 1 wereconcentrated using a rotary evaporator, and 30 ml of heptane was addedthereto, followed by reconcentration to dryness. TFA which was includedduring the separation operation was removed. The result of weightmeasurement showed the obtained D3G fraction was 1.51 g and the C3Gfraction was 0.98 g.

The D3G fraction and the C3G fraction were separately dissolved in 5%hydrochloric acid/95% methanol, and were then allowed to stand at 5° C.for 24 hours to perform crystallization. Solid liquid separation wascarried out using the Kiriyama funnel and No. 2 filter paper (Whatman),and washing with a small amount of acetone was carried out, followed bydrying to obtain precipitates. Both of the obtained precipitates wereobserved under a polarization microscope, and polarization of light wasobserved. This indicated that they were in crystal forms. The yield ofcrystalline D3G hydrochloride was 1.06 g and that of crystalline C3Ghydrochloride was 0.59 g.

The structures of the crystalline D3G hydrochloride and the crystallineC3G hydrochloride obtained were determined by NMR. The structures ofthese two types of anthocyanins were consistent with the spectrum datawhich have been already reported.

Other physical properties of the crystalline D3G hydrochloride and thecrystalline C3G hydrochloride are as follows.

Crystalline D3G hydrochloride:

Melting point based on thermoanalysis: 258° C. Uvλ max(ε): 517 nm(27500) FAB-MS m/z: M⁺:465

Compositional formula: C₂₁H₂₁O₁₂Cl•0.5H₂O Elementary analysis: C H ClMeasured values: 48.00 4.50 6.80 Calculated values 47.78 4.58 6.72Crystalline C3G hydrochloride:

Melting point based on thermoanalysis: 245° C. Uvλ max(ε): 510 nm(26300) FAB-MS m/z: M⁺:449

Compositional formula: C₂₁H₂₁O₁₁Cl•0.5H₂O Elementary analysis: C H ClMeasured values: 48.80 4.70 6.90 Calculated values: 49.57 4.73 6.93

EXAMPLE 3 Production of Purified delphinidin-3-O-rutinoside and Purifiedcyanidin-3-O-rutinoside Using Almond-Derived β-glucosidase

The powder (3.42 g) obtained in Reference Example 1 was dissolved in 1liter of 50 mM acetate buffer (pH 3.5) to prepare an anthocyaninsubstrate solution.

Separately, 208 g of almond-derived 0-glucosidase (SIGMA) was dissolvedin 1 liter of 50 mM acetate buffer (pH 3.5) to prepare an enzymesolution (corresponding to 500 U/ml). The substrate solution (1 liter)was heated at 40° C. for 10 minutes to stabilize the temperature.Thereafter, 1 liter of enzyme solution was added and thoroughly stirredto initiate the reaction. Sixty minutes later, 2 liters of 0.3Nhydrochloric acid was added to terminate the reaction.

Subsequently, 4 liters of reaction mixture, in which the reaction wasterminated, was treated by the adsorption of an ion exchange resin inthe same manner as described in Example 1, and further purified by HPLCusing an ODS-silica gel column. Thus, a D3R fraction with an R.T. of 69to 96 minutes and a C3R fraction with an R.T. of 144 to 174 minutes wereobtained.

These fractions were analyzed under the HPLC analysis conditions asdescribed in Example 1 (the R.T.s of the sample D3R and C3R were 12.63minutes and 18.19 minutes, respectively). The results showed that theD3R fraction and the C3R fraction had a single peak. Thus, purifieddelphinidin-3-O-rutinoside and purified cyanidin-3-O-rutinoside having apurity level of 99% or higher could be obtained.

The anthocyanin contents in the substrate solution before the reactionand that in the solution after the reaction which were measured by HPLCare shown in Table 2. According to Table 2, only anthocyanidin glucosidewas degraded and most of anthocyanidin rutinoside remained undegraded.This indicates that a reaction solution having an optimal compositionfor purification was obtained.

TABLE 2 Change in anthocyanin content D3G D3R C3G C3R Before reaction60.3 mg 231 mg 19.8 mg 171 mg After reaction  5.6 mg 207 mg  0.0 mg 162mg

EXAMPLE 4 Production of Crystalline delphinidin-3-O-rutinosideHydrochloride Hydrate and Crystalline Cyanidin-3-O-rutinosideHydrochloride Hydrate

Precipitates were obtained through the crystallization process as withExample 2. Both the obtained precipitates were observed under apolarization microscope, and as a result, polarization of light wasobserved and they were found to be in crystal forms. The yield ofcrystalline C3R hydrochloride was 58 mg and that of crystalline D3Rhydrochloride was 88 mg.

The structures of the crystalline D3R hydrochloride and the crystallineC3R hydrochloride obtained were determined by NMR. The structures ofthese two types of anthocyanins were consistent with the spectrum datawhich have been already reported.

Physical properties of the crystalline D3R hydrochloride and thecrystalline C3R hydrochloride are as follows.

Crystalline D3R hydrochloride:

Melting point based on thermoanalysis: 224° C. Uvλ max(ε): 520 nm(27800) FAB-MS m/z: M⁺:611

Compositional formula: C₂₇H₃₁O₁₆Cl•1.5H₂O Elementary analysis: C H ClMeasured values: 45.80 5.30 5.20 Calculated value: 45.86 5.38 4.98

Crystalline C3R hydrochloride:

Melting point based on thermoanalysis: 214 to 226° C. Uvλ max(ε): 512 nm(27400) FAB-MS m/z: M⁺:595

Compositional formula: C₂₇H₃₁O₁₅Cl•0.5H₂O Elementary analysis: C H ClMeasured values: 50.00 5.30 5.30 Calculated values: 49.97 5.13 5.46

COMPARATIVE EXAMPLE 1 Enzyme Reaction Using Commercially AvailableAnthocyanase

The powder (342 mg) obtained in Reference Example 1 was dissolved in 100ml of 50 mM acetate buffer (pH 3.5) to prepare an anthocyanin substratesolution.

Separately, CYTOLASE PCL5 (tradename, GIST-brocades), which is a type ofrepresentative anthocyanase used in the fruit juice industry, wasdiluted to 10-fold with a 50 mM acetate buffer (pH 3.5) to obtain anenzyme solution.

The substrate solution (2 ml) was heated at 40° C. for 10 minutes tostabilize the temperature. Thereafter, 2 ml of enzyme solution was addedand thoroughly stirred to initiate the reaction. Fifteen minutes later,200 μl of a reaction solution was sampled, and 200 μl of 0.3Nhydrochloric acid was added to terminate the reaction.

The anthocyanin compositions before the reaction and after the reactionwere measured by HPLC. The anthocyanin contents and compositions beforeand after the reaction are shown in Table 3 below. Table 3 shows thatboth the anthocyanidin glucoside and the anthocyanidin rutinoside weredegraded, indicating the composition was unsuitable for purification ofanthocyanin glycoside.

TABLE 3 Change in anthocyanin content D3G D3R C3G C3R Before reaction0.60 mg 2.31 mg 0.20 mg 1.71 mg After reaction 0.23 mg 0.09 mg 0.26 mg0.04 mg

INDUSTRIAL APPLICABILITY

The present invention enabled the production of highly purifiedanthocyanin and crystalline anthocyanin salt through purification fromnatural products. The thus produced crystalline anthocyanin salt did notexhibit hygroscopicity and was stable.

This specification includes part or all of the contents as disclosed inthe specification of Japanese Patent Application No. 2000-276540, whichis a priority document of the present application. All publications,patents and patent applications cited herein are incorporated herein byreference in their entirety.

1. A process for producing purified anthocyanidin glucoside, whereinrhamnosidase is contacted with an anthocyanin composition containing atleast one anthocyanidin rutinoside to convert the anthocyanidinrutinoside into anthocyanidin glucoside, which is then isolated andpurified.
 2. The process for producing purified anthocyanidin glucosideaccording to claim 1, wherein the anthocyanin composition is fruit juiceobtained from at least one member selected from black currant, fig,coffee, banana, and blackberry and/or an anthocyanin concentrateobtained from wild rice (Zizania aquatica Linn.) or colocasia.
 3. Theprocess for producing purified anthocyanidin glucoside according toclaim 1, wherein the rhamnosidase is hesperidinase or naringinase.
 4. Aprocess for producing crystalline anthocyanidin-3-O-glucosidehydrochloride hydrate comprising: a) contacting rhamnosidase with ananthocyanin composition containing at least one anthocyanidin rutinosideto convert the anthocyanidin rutinoside into anthocyanidin glucoside; b)purifying the anthocyanidin glucoside to obtain anthocyanidin glucosideof 99% or higher purity; and c) crystallizing the anthocyanidinglucoside of 99% or higher purity using a mixed solvent of hydrochloricacid/alcohol system to obtain crystalline anthocyanidin-3-O-glucosidehydrochloride hydrate.
 5. The process for producing crystallineanthocyanidin-3-O-glucoside hydrochloride hydrate according to claim 4,wherein the purification process according to step b) in claim 4 iscarried out by ion exchange adsorption chromatography and/or HPLC. 6.The process for producing crystalline anthocyanidin-3-O-glucosidehydrochloride hydrate according to claim 4, wherein the mixed solvent ofhydrochloric acid/alcohol system is composed of 5% (v/v) hydrochloricacid /95% (v/v) methanol.
 7. The process for producing purifiedanthocyanidin glucoside according to claim 2, wherein the rhamnosidaseis hesperidinase or naringinase.